The project entitled "Immune recognition of glycolipids" fits into a collaborative Program Project by addressing biochemical and structural questions that complement biological studies led by Dr. Bendelac and by using extensively chemical compounds developed in collaboration with Dr. Savage. Specific Aim 1: Define the nature of lipids and glycolipids interactions with CD1. State of the art biophysical methods will be used to measure the interaction of murine CD1d with a large number of phospholipids and glycolipids. Rules for binding to CD1 will be established for alphaGC using variants of alphaGC. Binding constants will be correlated to biological functional assays. Binding of synthetic ligands and natural ligands bound to CD1d will also be studied and quantified by mass spectrometry. Tetrameric and multimeric recombinant Valpha14 T cell receptors will be used to detect loaded CD1 molecules in vivo. Specific Aim 2: Define the rules that govern T cell recognition of CD1-glycolipids complexes. Recombinant Valpha14 T cell receptors will be expressed in a fly expression system as well as in bacteria. Classical biophysical techniques such as surface plasmon resonance will be used to measure the affinity of these receptors for CD1-glycolipid complexes. Contribution of CD1 residues and contribution of sugar moieties to binding will be studied by mutagenesis of CD1 and usage of alphaGC variants. Alanine scanning of the CDR regions of both alpha and beta chain will help understand the orientation of recognition. This point will be further investigated by using photoactivable variants of alphaGC. The recombinant receptors will also be used to raise anti-Valpha14 antibodies. Specific Aim 3: Additional structural studies. Aims 1 and 2 will provide a wealth of structural information on the binding of glycolipids to CD1 and their recognition by alpha/beta T cell receptors. However, the direct visualization of these structural features will be attempted by 2- and 3-dimensional crystallography. We will attempt the crystallization single lipids, of CD1 loaded with the crystallization of Valpha14 T cell receptors and Valpha14 TCP/CD1-alphaGC complexes. We know from experience that this approach is highly unpredictable but we also know that it is unlikely that a sustained effort won't deliver some results. This approach has to be tried in the context of Aims 1 and 2.